Implementation of clonal tree production is a major step that will be taken by the forest products industry in coming years. Clonal deployment of advance generation selections is expected to increase forest productivity by some 10% to 20% and increase uniformity of the fiber output (Libby et al., For. Chron. 60:145-149 (1984)). Significant economic and biological barriers exist to large-scale clonal propagation and plantation of softwood trees (Stelzer et al., Can. J. For. Res. 27:442-446 (1997)). For many softwood trees including loblolly pine, somatic embryogenesis is the most promising method to overcome these barriers (Cheliak et al., Can. J. For. Res. 20:452-463 (1990)). Somatic embryogenesis is widely used in a variety of plant species to produce multiple copies of genetically identical organisms. Somatic embryogenesis is becoming the method of choice for clonal propagation of spruce. However, for loblolly pine, the most important species in the United States, somatic embryogenesis has lagged behind.
In some species, somatic embryogenesis is used to propagate desirable plant genotypes. In many crop species, somatic embryogenesis is used to propagate whole transformed plants from tissues that have been genetically altered. Somatic embryogenesis protocols have been developed for the reproduction of conifers. For example, U.S. Pat. Nos. 4,957,866, 5,034,326, 5,036,007, 5,236,841, 5,413,930, 5,491,090, and 5,506,136 (the disclosures of which herein incorporated by reference), describe various methods and media for conifer embryogenesis.
Somatic embryogenesis is a multi-step process by which an individual plant is clonally propagated. Tissue from the parent plant is induced to form embryos. Subsequent culturing steps are then performed to induce the embryos to mature into plantlets. These small plants are capable of growing into mature plants, each of which is genetically identical to the parent. Using this process, an individual plant with a desirable genotype can be efficiently reproduced
In conifers, somatic embryogenesis begins with “initiation,” the initial formation of embryogenic cultures. Embryogenic cultures contain one or more immature somatic embryos. Initiation is traditionally followed by a maintenance and multiplication phase in which large numbers of clones of the initiated somatic embryo(s) are produced. The embryos produced in the maintenance and multiplication step(s) are then further cultured on a development or maturation media. This development medium induces the immature embryos to mature into late-stage embryos capable of germination. These mature embryos are often placed on a germination medium where they germinate to form plantlets. The plantlets can be grown and acclimated to the point that they are capable of being planted in soil. Subsequently these small plantlets will grow into mature trees.
Many conifer species contain recalcitrant genotypes not readily regenerated. The commercially important loblolly pine, for example, is generally difficult to propagate by somatic embryogenesis (Becwar et al., Can. J. For. Res. 20:810 (1990), Jain et. al., Plant Sci. 65:233-241 (1989)). Further, genetic transformation techniques used to generate transgenic conifers, such as Agrobacterium-mediated gene transfer, electroporation, and particle bombardment, can damage plant cells. Damaged cells are less likely to regenerate into whole plants. Thus, there is a need in the art for methods and compositions that improve the efficiency of somatic embryogenesis, including somatic embryogenesis in conifers.
Initiation of Embryogenic Cultures
Somatic embryogenesis begins with the “initiation” step. Initiation starts with the selection of a suitable explant, that is any plant cell, tissue or organ capable of forming an embryogenic culture. A typical explant in conifer somatic embryogenesis is the megagametophyte, also called the ovule or the female gametophyte, which is extracted from a pollinated female cone and which may contain single or multiple zygotic seed embryos. One or more cells of the explant are then induced to proliferate into a tissue mass containing at least one early stage somatic embryo. The successful establishment of such a culture is known as initiation.
The cultures can be initiated from several types of explants. Most commonly, conifers embryogenic cultures are initiated from zygotic embryos or zygotic embryogenic tissues found in seeds. When intact conifer megagametophytes are used as the explant, the somatic embryogenesis process comprises a distinct step called extrusion. Extrusion is the process in which a mass of embryogenic tissue is extruded from the micropylar end of the megagametophyte when it is placed on or in suitable culture media.
Successfully initiating embryogenic cultures require proper medium and culturing conditions. In conifers, an embryogenic culture is successfully initiated when the zygotic embryo or zygotic embryogenic tissue mass, which has been either extruded or physically removed from a megagametophyte, undergoes division and proliferation. A successfully initiated culture consists of a whitish translucent mucilaginous tissue mass that contains pre-embryonal cells, filamentous suspensor-like cells, and early stage somatic embryos. In a successfully initiated culture, new somatic embryos can often be seen growing directly from older zygotic embryos. Visualization of initiation is aided by the fact that zygotic embryos, as well as extruded tissues, often become brown while initiated tissues are whiter and more translucent. Initiated cultures contain from one to dozens of somatic embryos. In culture, initiation frequency is scored for each dish by counting the number of explants that had successfully initiated embryogenic cultures. Initiation is considered successful when at least one somatic embryo is visible. The appearance of at least two somatic embryos provides a useful confirmation of successful initiation.
Maintenance and Multiplication of Embryogenic Cultures
In conifer somatic embryogenesis, initiation is generally followed by one or more “maintenance and multiplication” steps. Some protocols consist of several maintenance and multiplication steps, each with its own media and culturing conditions. Other protocols utilize a single maintenance and multiplication step. “Maintenance” refers to the preservation of cultures by keeping them alive and viable through continuous growth. “Multiplication” refers to the proliferation of such cultures to provide numerous somatic embryos. Regardless of whether the steps are separate or combined into a single culturing step, this phase of somatic embryogenesis requires that previously initiated embryogenic cultures survive and proliferate, continuously producing viable immature somatic embryos. This requires the proper media and culture conditions.
Various methods of maintaining and multiplying cultures have been described. In some protocols, initiated cultures are grown using a single medium composition prior to transfer to a development and maturation media. For example, U.S. Pat. No. 5,563,061 describes a method for conifer somatic embryogenesis wherein initiated embryogenic cultures are transferred to a single maintenance and multiplication medium where they are cultured to increase the number and size of the embryos. After culturing on this maintenance and multiplication media, the somatic embryos are transferred to maturation and development media. Notably, however, the development, maturation, and germination steps of somatic embryogenesis are not 100% efficient. In order for this protocol to be used effectively, a very large number of somatic embryos must be produced initially.
U.S. Pat. No. 5,491,090 describes methods of somatic embryogenesis where initiated cultures are transferred to a liquid maintenance culture. This patent additionally describes a protocol where embryogenic cultures are initiated on a first media, transferred to a second media, and thereafter transferred to a liquid media for rapid multiplication. The use of a liquid culture is especially advantageous in that handling liquid cultures is much less labor-intensive than manipulating cultures on solid media. This ease of handling makes liquid culture practical for a large-scale production of seedlings via somatic embryogenesis. Very large amounts of somatic embryos can be efficiently produced in liquid cultures, with cultures multiplying as much as 2-6 times weekly. However, the inventors have observed that tissue transferred to liquid culture often does not survive and proliferate. Thus, there is a need in the art for methods that will increase the efficiency of embryogenic culture multiplication using liquid media.
The success of the maintenance and multiplication step is vital for the ultimate generation of plantlets. Embryogenic cultures, once successfully initiated, must proliferate and supply sufficient numbers of somatic embryos such that a reasonable number will ultimately be converted to plantlets. There is a need in the art for methods which improve the growth of embryogenic cultures such that large numbers of somatic embryos may be produced. Further, methods which improve the growth of embryogenic cultures will save time and reduce production costs by more rapidly producing the desired numbers of embryos. Additionally, efficient methods of maintaining and multiplying embryogenic cultures can reduce the number of culturing steps, greatly reducing the time and expenses of the propagation process.